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PubMed1996

Negative allosteric modulation of wild-type and mutant AMPA receptors by GYKI 53655.

Benzothiadiazides such as cyclothiazide potentiate alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor responses, whereas 2,3-benzodiazepines such as 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8-methylenedioxy-3,4 -dihydro- 5H-2,3-benzodiazepine (GYKI 53655) act as noncompetitive antagonists; both drugs act through allosteric modulation. Controversy exists as to whether cyclothiazide and GYKI 53655 act at a common site. Recent mutational analysis has led to the identification of a serine residue in flip splice variants that is critical for directing the interaction of cyclothiazide with AMPA receptors. We tested whether the mutation of this residue to glutamine, which abolishes potentiation by cyclothiazide, can in addition block antagonism by 2,3-benzodiazepines, as would be predicted for action at a common site. We found that the S to Q mutation does not alter antagonism by 2,3-benzodiazepines, suggesting that the molecular determinants directing the interaction between GYKI 53655 and AMPA receptors are not identical to those controlling sensitivity to cyclothiazide. Additional support for this was obtained from analysis of the responses of AMPA receptor flip/flop splice variants, which, despite differences in equilibrium desensitization and sensitivity to cyclothiazide, show only small differences in sensitivity to 2,3-benzodiazepines. Furthermore, introduction of the flip exon from GluRA into GluR6, conferred sensitivity to cyclothiazide but did not increase sensitivity to 2,3-benzodiazepines. Of interest, experiments with native AMPA receptors generated from hippocampal and forebrain poly(A)+ mRNA revealed greater sensitivity to 2,3-benzodiazepines than receptors generated by expression of recombinant AMPA receptors, possibly indicating the existence of an unidentified accessory protein or novel receptor subunit.

Allosteric Regulation↗
PubMed1993

Substitution of glutamic acid for alanine 1135 in the putative "catalytic loop" of the tyrosine kinase domain of the human insulin receptor. A mutation that impairs proteolytic processing into subunits and inhibits receptor tyrosine kinase activity.

The intracellular domain of the insulin receptor possesses activity as a tyrosine-specific protein kinase which is stimulated by insulin binding to the extracellular domain of the receptor. We have identified a patient with a genetic form of insulin resistance who is heterozygous for a mutation substituting Glu for Ala1135 in the putative "catalytic loop" of the tyrosine kinase domain of the receptor. In this investigation, the Glu1135 mutant receptor was expressed by transfection of mutant cDNA into NIH-3T3 cells. Like previously described mutations in the tyrosine kinase domain, the Glu1135 mutation impairs receptor tyrosine kinase activity and inhibits the ability of insulin to stimulate thymidine incorporation and receptor endocytosis. These data support the hypothesis that the receptor tyrosine kinase activity plays a necessary role in the ability of the receptor to mediate insulin action in vitro and in vivo. However, unlike previously described mutations in the intracellular domain of the receptor, the Glu1135 mutation impairs proteolytic cleavage of the proreceptor into separate subunits and impairs the transport of the receptor to the cell surface. These latter defects provide an explanation for the decrease in the number of receptors on the cell surface observed in the patient's circulating monocytes despite the fact that the mutant receptor is resistant to endocytosis and insulin-induced down-regulation.

3T3 Cells↗
PubMed1994

Resistance to thyroid hormone in children.

The genetic etiology of thyroid hormone resistance syndromes is now well established. Two clinical variants, generalized resistance to thyroid hormone (GRTH) and selective pituitary resistance to thyroid hormone (PRTH), are, in most cases, caused by heterozygous mutations in the ligand-binding domain of the c-erbA beta thyroid hormone receptor gene. No human mutations have yet been described in the other related receptor gene, c-erbA alpha. In resistant patients, the mutant beta receptors act as dominant negative proteins and inhibit function of the normal beta receptor (expressed from one allele) and the normal alpha receptor (expressed from two alleles). Patients homozygous for a dominant negative allele (the Bercu patient) and without any beta receptor (the Refetoff patient) have been described. Patients with GRTH and PRTH both present with elevated free thyroxine and triiodothyronine and inappropriately normal thyroid-stimulating hormone, but the former patients are clinically euthyroid, whereas the latter patients have symptoms and signs of hyperthyroidism. However, in some cases, different patients who have been classified as having GRTH and PRTH have been found to have identical beta mutations. A recent study of the level of pituitary resistance in a large kindred with GRTH (ARG-320-HIS mutation) indicated a contributory gene in the regulation of thyroid hormone action. Relative overexpression of the mutant PRO-453-HIS receptor at the level of messenger RNA in patient fibroblasts (kindred A) was associated with short stature. Finally, an ARG-316-HIS mutation (kindred G-H) was associated with relatively weak dominant negative activity and perturbed DNA-binding properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Attention Deficit Disorder with Hyperactivity↗
PubMed2003

Effects of disruption of heat shock genes on susceptibility of Escherichia coli to fluoroquinolones.

BACKGROUND: It is well known that expression of certain bacterial genes responds rapidly to such stimuli as exposure to toxic chemicals and physical agents. It is generally believed that the proteins encoded in these genes are important for successful survival of the organism under the hostile conditions. Analogously, the proteins induced in bacterial cells exposed to antibiotics are believed to affect the organisms' susceptibility to these agents. RESULTS: We demonstrated that Escherichia coli cells exposed to levofloxacin (LVFX), a fluoroquinolone (FQ), induce the syntheses of heat shock proteins and RecA. To examine whether the heat shock proteins affect the bactericidal action of FQs, we constructed E. coli strains with mutations in various heat shock genes and tested their susceptibility to FQs. Mutations in dnaK, groEL, and lon increased this susceptibility; the lon mutant exhibited the greatest effects. The increased susceptibility of the lon mutant was corroborated by experiments in which the gene encoding the cell division inhibitor, SulA, was subsequently disrupted. SulA is induced by the SOS response and degraded by the Lon protease. The findings suggest that the hypersusceptibility of the lon mutant to FQs could be due to abnormally high levels of SulA protein resulting from the depletion of Lon and the continuous induction of the SOS response in the presence of FQs. CONCLUSION: The present results show that the bactericidal action of FQs is moderately affected by the DnaK and GroEL chaperones and strongly affected by the Lon protease. FQs have contributed successfully to the treatment of various bacterial infections, but their widespread use and often misuse, coupled with emerging resistance, have gradually compromised their utility. Our results suggest that agents capable of inhibiting the Lon protease have potential for combination therapy with FQs.

ATP-Dependent Proteases↗
PubMed1994

Comparison of the sites of reaction of three polycyclic aromatic hydrocarbons in the supF gene.

The distribution of hydrocarbon-DNA adducts through the supF gene in plasmid pS189 was examined using the polymerase arrest assay. For three hydrocarbon dihydrodiol epoxides, derived from 5-methylchrysene, 7-methylbenz[a]anthracene, and benzo[a]pyrene, that exhibit a preference for reaction with guanine residues in DNA, polymerase arrest spectra were similar but not identical. For each agent, guanines in different sequence contexts exhibited varying reactivities and each specific guanine did not necessarily respond to each agent in the same fashion. Thus, sequence context together with the individual dihydrodiol epoxide's chemical and physical properties all play a role in determining sites and extents of reaction within a specific gene. The polymerase arrest data were not predictive of the known sites of mutation hotspots for these dihydrodiol epoxides in the supF gene indicating that further action upon the adducted DNA by repair systems is probably necessary to determine which specific chemical adducts will ultimately give rise to mutation.

Base Sequence↗
PubMed2004

Reverse transcriptase activity of hepatitis B virus (HBV) DNA polymerase within core capsid: interaction with deoxynucleoside triphosphates and anti-HBV L-deoxynucleoside analog triphosphates.

The use of L(-)SddC [beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC)] for the treatment of Herpes B virus (HBV) infection is hindered by the emergence of drug-resistance associated with the L526M, L550V, and L526M/M550V mutations of the viral DNA polymerase (DP). The interactions of the anti-HBV compounds 2',3'-dideoxy-2',3'-didehydro-beta-L(-)-5-fluorode-oxycytidine and 2'-fluoro-5-methyl-beta-L-arabinofuranosyluracil triphosphate with HBV DP and its L(-)SddC-associated mutants have not been studied. The e antigen-negative variant of HBV associated with the G1896A mutation in the precore region has a high prevalence. Its effect on HBV DP is unclear. Because HBV DNA synthesis occurs in the nucleocapsid, we examined the kinetics of the reverse transcriptase activity from wild-type (wt) and mutated DPs with the wt or G1896A-mutated RNA template in the nucleocapsid. The effects of this template mutation on the activities of these L-nucleoside triphosphates were also examined. Results indicated that these DP mutations increased the Km values of deoxy-NTPs and decreased the efficiencies (Vmax/Km) of DPs. The additional L526M mutation increased the efficiency of the M550V-mutated DP but no more than that of the L526M-mutated DP. The G1896A mutation had impacts on the interactions between different DPs and deoxy-NTPs, except dCTP. It also had different impacts on the actions of the L-nucleoside triphosphates toward DPs. The L526M and M550V mutations caused a greater decrease in the Vmax using the wt RNA template compared with the G1896A-mutated template. The L526M, M550V, and L526M/M550V mutations caused varying degrees of resistance to the different M-nucleoside triphosphates.

Animals↗
PubMed2000

Mice with a targeted mutation in the thyroid hormone beta receptor gene exhibit impaired growth and resistance to thyroid hormone.

Patients with mutations in the thyroid hormone receptor beta (TRbeta) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TRbeta in vivo, we generated mice with a targeted mutation in the TRbeta gene (TRbetaPV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre/loxP system. Mice expressing a single PV allele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary-thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TRbeta gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TRbetaPV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TRbeta in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients.

Alleles↗
PubMed1972

Membrane mutation associated with sensitivity to acriflavine in Escherichia coli.

The role of plasma membrane on the acriflavine sensitivity of Escherichia coli was studied. (14)C-uracil incorporation into ribonucleic acid fraction by spheroplasts was more sensitive to acriflavine in the acriflavine-sensitive strain (genotype acrA) than in the acriflavine-resistant (genotype acrA(+)) strain. There was no difference between two types of cells in the response to osmotic shock, phage sensitivity, and other treatments used to investigate the structure and stability of cell wall. Differences in the electron-microscopic figures between acrA and acrA(+) cells was found in the plasma membrane, surface area just below the membrane, and ribosomal aggregation, when cells were treated with acriflavine. It is concluded that a primary site of acriflavine action is on the plasma membrane, and the acrA mutation is mediated by it. On the basis of the present results, it is evident that differences in the acriflavine binding and the sensitivity to phenethyl alcohol and sodium dodecyl sulfate between the acrA and acrA(+) strains, previously reported, are attributable to a structural difference in the plasma membrane between the two strains.

Acridines↗
PubMed2001

Early development of the gubernaculum and cremaster sac in estrogen receptor knockout mice.

AIM exogenous estrogen causes gubernacular atrophy and cryptorchidism in fetal rodents. Mice with an estrogen receptor-alpha (ERalpha) disrupted gene mutation (alphaERKO) were studied to determine whether ablation of endogenous estrogen action, through ERalpha, had an effect on gubernacular development. Serial sagittal sections were made of the pelvis in fetal and day 7 postnatal wild-type and alphaERKO mice with the estrogen receptor-alpha "knockout" gene mutation. Wild-type (n = 24), heterozygote (n = 13) and alphaERKO mice (n = 12) were sacrificed at 16, 17 and 18 days fetal life and at 7 days postnatally. The size of the gubernaculum, cremaster muscle, cremaster sac, and the width of the sac at both ends in day 7 mice were quantitated by computer analysis. Visually and statistically the ERKO mice could not be separated from the wild-type mice during fetal life. At day 7 postnatally, a thicker cremaster sac was noted morphologically, and also a statistically significant difference was seen in the width of the cremaster sac at the sac's tip. Sac area, cremaster muscle area and the width of the sac at the sac's end did not differ significantly. Overall there is minimal phenotypic change observed in the alphaERKO mouse compared to wild-type at the early developmental stages investigated. However, at postnatal day 7, there is a difference in the width of the cremasteric sac tip. This suggests that the effect of ERalpha, and thus signaling on the developing gubernaculum, occurs late in development. Alternatively, an action from the recently discovered ERbeta may be involved. Exploration of a betaERKO and the double knock-out alphaERKO/betaERKO mouse should be informative in evaluating the effect of endogenous estrogens in gubernacular development.

Animals↗
PubMed1976

Genetic evidence of ribosomal antisuppressors in Podospora anserina.

Antisuppressors were screened for with the help of informational suppressors in Podospores anserina. Four mutations in the AS1 locus and two in the AS2 locus were isolated, using allele non specific suppressors supposed to be ribosomal ambiguity mutations. Four mutations in the AS3 locus and 45 in the AS4 locus were obtained, using a nonsense (t-RNA like) suppressor. All antisuppressors are partially dominant. Most mutations in the AS4 locus are lethal. The four mutants at the AS3 locus and 6 out of the 8 viable mutants at the AS4 locus are cold sensitive. Phenotypic properties and action spectra of the antisuppressors suggest that they are restrictive ribosomal mutations.

Ascomycota↗
PubMed2001

Mechanisms for evolving hypervariability: the case of conopeptides.

Hypervariability is a prominent feature of large gene families that mediate interactions between organisms, such as venom-derived toxins or immunoglobulins. In order to study mechanisms for evolution of hypervariability, we examined an EST-generated assemblage of 170 distinct conopeptide sequences from the venoms of five species of marine Conus snails. These sequences were assigned to eight gene families, defined by conserved elements in the signal domain and untranslated regions. Order-of-magnitude differences were observed in the expression levels of individual conopeptides, with five to seven transcripts typically comprising over 50% of the sequenced clones in a given species. The conopeptide precursor alignments revealed four striking features peculiar to the mature peptide domain: (1) an accelerated rate of nucleotide substitution, (2) a bias for transversions over transitions in nucleotide substitutions, (3) a position-specific conservation of cysteine codons within the hypervariable region, and (4) a preponderance of nonsynonymous substitutions over synonymous substitutions. We propose that the first three observations argue for a mutator mechanism targeted to mature domains in conopeptide genes, combining a protective activity specific for cysteine codons and a mutagenic polymerase that exhibits transversion bias, such as DNA polymerase V. The high D:(n)/D:(s) ratio is consistent with positive or diversifying selection, and further analyses by intraspecific/interspecific gene tree contingency tests weakly support recent diversifying selection in the evolution of conopeptides. Since only the most highly expressed transcripts segregate in gene trees according to the feeding specificity of the species, diversifying selection might be acting primarily on these sequences. The combination of a targeted mutator mechanism to generate high variability with the subsequent action of diversifying selection on highly expressed variants might explain both the hypervariability of conopeptides and the large number of unique sequences per species.

Animals↗
PubMed2002

Neutrophil elastase enzymatically antagonizes the in vitro action of G-CSF: implications for the regulation of granulopoiesis.

There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3, elastase, azurocidin, and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells, elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF.

Adult↗
PubMed2001

Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1.

The hormone auxin is transported in plants through the combined actions of diffusion and specific auxin influx and efflux carriers. In contrast to auxin efflux, for which there are well documented inhibitors, understanding the developmental roles of carrier-mediated auxin influx has been hampered by the absence of specific competitive inhibitors. However, several molecules that inhibit auxin influx in cultured cells have been described recently. The physiological effects of two of these novel influx carrier inhibitors, 1-naphthoxyacetic acid (1-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA), have been investigated in intact seedlings and tissue segments using classical and new auxin transport bioassays. Both molecules do disrupt root gravitropism, which is a developmental process requiring rapid auxin redistribution. Furthermore, the auxin-insensitive and agravitropic root-growth characteristics of aux1 plants were phenocopied by 1-NOA and CHPAA. Similarly, the agravitropic phenotype of inhibitor-treated seedlings was rescued by the auxin 1-naphthaleneacetic acid, but not by 2,4-dichlorophenoxyacetic acid, again resembling the relative abilities of these two auxins to rescue the phenotype of aux1. Further investigations have shown that none of these compounds block polar auxin transport, and that CHPAA exhibits some auxin-like activity at high concentrations. Whilst results indicate that 1-NOA and CHPAA represent useful tools for physiological studies addressing the role of auxin influx in planta, 1-NOA is likely to prove the more useful of the two compounds.

Arabidopsis↗
PubMed1999

Mutations in the yeast two pore K+ channel YKC1 identify functional differences between the pore domains.

The K+ channel of Saccharomyces cerevisiae encoded by the YKC1 gene includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore. Gating of the channel is promoted by membrane depolarisation and is regulated by the extracellular K+ concentration ([K+]o) both in the yeast and when expressed in Xenopus oocytes. Our previous work showed that substitutions of equivalent residues L293 and A428 within the pore-loops had qualitatively similar effects on both the [K+]o-sensitivity of channel gating and its voltage-dependence. Here, we report that mutations of equivalent residues N275 and N410, N-terminal from the K+ channel signature sequences of the two pores, have very different actions on channel gating and, in this case, are without effect on its voltage-sensitivity. The mutation N410D slowed current activation in a [K+]o-dependent manner and it accelerated deactivation, but without significant effect on the apparent affinity for K+. The N275D mutant, by contrast, had little effect on the [K+]o-sensitivity for activation and it greatly altered the. [K+]o-dependence of current deactivation. Neither mutant affected the voltage-dependence of the steady-state current nor the ability for other alkali cations to substitute for K+ in regulating gating. The double mutant N410D-N275D showed characteristics of N410D in the [K+]o-sensitivity of current activation and of N275D in the [K+]o-sensitivity of deactivation, suggesting that little interaction occurs between pore domains with mutations at these sites. The results indicate that the two pore domains are not functionally equivalent and they suggest that the regulation of gating by external K+ is mediated by K+ binding at two physically distinct sites with different actions.

Animals↗
PubMed2005

Functional effects of KCNJ11 mutations causing neonatal diabetes: enhanced activation by MgATP.

Recent studies have shown that heterozygous mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium (K(ATP)) channel, cause permanent neonatal diabetes either alone (R201C, R201H) or in association with developmental delay, muscle weakness and epilepsy (V59G,V59M). Functional analysis in the absence of Mg2+, to isolate the inhibitory effects of ATP on Kir6.2, showed that both types of mutation reduce channel inhibition by ATP. However, in pancreatic beta-cells, K(ATP) channel activity is governed by the balance between ATP inhibition via Kir6.2 and Mg-nucleotide stimulation mediated by an auxiliary subunit, the sulphonylurea receptor SUR1. We therefore studied the MgATP sensitivity of KCNJ11 mutant K(ATP) channels expressed in Xenopus oocytes. In contrast to wild-type channels, Mg2+ dramatically reduced the ATP sensitivity of heterozygous R201C, R201H, V59M and V59G channels. This effect was predominantly mediated via the nucleotide-binding domains of SUR1 and resulted from an enhanced stimulatory action of MgATP. Our results therefore demonstrate that KCNJ11 mutations increase the current magnitude of heterozygous K(ATP) channels in two ways: by increasing MgATP activation and by decreasing ATP inhibition. They further show that the fraction of unblocked K(ATP) current at physiological MgATP concentrations correlates with the severity of the clinical phenotype.

ATP-Binding Cassette Transporters↗
PubMed1975

Cell cycle-dependent induction of mutations along a yeast chromosome.

The relation between DNA replication and the action of the mutagen N-methyl-N'-nitro-N-nitroso-guanidine has been studied in Saccharomyces cerevisiae. The frequenceis of reversion to prototrophy of six auxotrophic markers located along one arm of chromosome VII were examined as a function of the vegetative cell cycle. Exponentially growing cells were treated with nitrosoguanidine and then separated by zonal rotor centrifugation into fractions equivalent to stages in the cell cycle. The frequency of reversion for five of the six markers is greatest during the period of DNA replication. Each marker has a single point of maximum reversion, approximately 10-fold greater than the frequency observed at other points in the cell cycle. For any one marker the effect of nitrosoguanidine is restricted to an interval shorter than the period of DNA replication. The two markers most distant from each other, ade5 and leul, both have their highest reversion frequency early during DNA replication. The peak reversion frequency for lys5 is somewhat later, while the peaks for tyr3 and trp5 occur near the end of DNA replication. The results indicate that nitrosoguanidine acts primarily during DNA replication and that different markers appear to be affected at different intervals during the DNA biosynthetic period. If nitrosoguanidine does act at the growing point of DNA replication, these observations indicate that the initiation of DNA replication occurs at specific times during the period of DNA synthesis and at specific initiation sites. Further, there must be more than one point of initiation of DNA replication on one arm of chromosome VII.

Cell Division↗
PubMed2003

PTEN decreases in vivo vascularization of experimental gliomas in spite of proangiogenic stimuli.

Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PTEN/MMAC. Additionally, these tumors are associated with (a) mutations in epidermal growth factor receptor (EGFR), leading to a pro-oncogenic constitutive activation, as well as amplification of its gene, and/or (b) mutations in p53, disrupting normal cellular homeostatic processes. Whereas PTEN/MMAC has been shown to possess antiangiogenic action, constitutively active EGFR or p53 gene defects have been associated with proangiogenic action. In this article, we asked if PTEN/MMAC gene transfer into human glioma cells that possess inactivating mutations of the PTEN/MMAC gene but also express either constitutively active EGFR (U87DeltaEGFR cells) or possess an inactivating mutation of p53 (U251 cells) still display inhibited angiogenesis in orthotopic and ectopic models of gliomas. Human glioma xenografts treated with PTEN/MMAC gene transfer exhibited significantly decreased vascularity both in an orthotopic and in an ectopic model. Taken in combination, these results provide strong evidence of PTEN/MMAC's role in regulating glioma angiogenesis even in the presence of strong proangiogenic signals provided by constitutive EGFR activation or p53 inactivation.

Animals↗